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Elimination of substrate inhibition of a β-N-acetyl-d-hexosaminidase by single site mutation

Paper ID Volume ID Publish Year Pages File Format Full-Text
34853 45052 2013 6 PDF Available
Title
Elimination of substrate inhibition of a β-N-acetyl-d-hexosaminidase by single site mutation
Abstract

Substrate inhibition hinders chitinolytic β-N-acetyl-d-hexosaminidases in producing N-acetyl-d-glucosamine (GlcNAc), the valuable chemical widely applied in medical and food industries. Here we focused on a promising chitinolytic enzyme, OfHex1 from the insect, Ostrinia furnacalis. By structural analysis of OfHex1, five residues nearby the active pocket including V327, E328, Y471, V484 and W490 were chosen and nine mutants including V327G, E328Q, E328A, Y471V, V484R, W490A, W490H, V327G/V484R/W490A and V327G/Y471V/W490H were constructed and recombinantly expressed in Pichia pastoris. The best-performing mutant, W490A, obtained by a higher yield of 5 mg/L, did not show substrate inhibition even when 5 mM of the substrates, (GlcNAc)2–4, were applied. The kcat/Km values for (GlcNAc)2–4 are 239.8, 111.3 and 79.8 s−1 mM−1, respectively. Besides, the pH stability of the mutant ranges from pH 4 to 11 and the thermal stability is up to 50 °C. This work suggests the W490A mutant might be an ideal biocatalyst for GlcNAc production from chitin.

► Substrate inhibition hinders enzymatic preparation of GlcNAc. ► The mutant W490A by site-directed mutagenesis is devoid of substrate inhibition. ► It is with high yield of 5 mg/L of culture medium. ► It exhibits promising activity with kcat/Km value of 239.8 s−1 mM−1 for (GlcNAc)2.

Keywords
GH20, glycosyl hydrolase family 20; GlcNAc, N-acetyl-d-glucosamine; HsHex, β-N-acetyl-d-hexosaminidase from human placenta; MU-β-GlcNAc, 4-methylumbelliferyl-N-acetyl-β-d-glucosaminide; OfHex1, β-N-acetyl-d-hexosaminidase from Ostrinia furnacalis; pNP-β-G
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Elimination of substrate inhibition of a β-N-acetyl-d-hexosaminidase by single site mutation
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 48, Issue 1, January 2013, Pages 103–108
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us